Base-Mediated Nitrophenyl Reductive Cyclization for the Synthesis of Hexahydro-2,6-methano-1-benzazocines

A Diels–Alder reaction leading to 4-nitrophenylcyclohexanones followed by a newly developed base-mediated reductive cyclization of the resulting ketone tethered to the nitrobenzene moiety gives access to the hexahydro-2,6-methano-1-benzazocine ring system present in several biologically interesting natural products such as aspernomine. The scope of the reaction was explored with eight substituted nitrobenzenes, obtaining yields of up to 87%. The highest cytotoxicity was observed in benzazocine 4h, bearing an enone moiety, which was active against eight cancer cell lines.

A wide variety of fungi produce morphological structures known as sclerotia that are key for long-term species survival and propagation. A study of the sclerotia of Aspergillus by Gloer's group led to the isolation of several biologically active secondary metabolites, 1 including the complex indole diterpenoid anominine, 2 most likely the parent structure from which the others are biogenetically derived. 3 In a previous study on this natural product, our group achieved the first total synthesis of anominine and established its absolute configuration. 4 Since then, a number of other products from this family have been synthesized. 5 Among these fungal metabolites, some are reported to have antiinsectan properties, whereas the cytotoxic aspernomine, 6 yet to be synthesized, has shown activity against the A549 lung carcinoma, MCF7 breast adenocarcinoma, and HT29 colon adenocarcinoma cell lines. Its anticancer properties could be attributable to the hexahydro-2,6-methano-1-benzazocine moiety, also present in the structurally related sespenine 3 or strychnochromine 7 (Figure 1).
Given the highly promising properties of these structures, the scarcity of precedents for their preparation indicates the synthetic challenge they pose. 8 In the present study, we targeted the synthesis of the hexahydro-2,6-methano-1-benzazocine ring system through a base-mediated intramolecular nitrophenyl reductive cyclization based on precedents reported by our group (Scheme 1a). In studies of the total synthesis of Strychnos indole alkaloids, attempts at an α-formylation of a nitrophenylketone scaffold using tris(dimethylamino)methane resulted in the accidental discovery of a cyclized product bearing a pyrrolobenzazocine framework (Scheme 1b). 9 In addition, in the context of the total synthesis of strychnine, endeavors to form a piperidine ring using propargylic and vinyl iodide precursors led to the unexpected formation of a bridged tetrahydroquinoline scaffold, albeit with low yields (Scheme 1c). 10 With these precedents in mind, the cyclization of methyl-1-(2nitrophenyl)-4-oxocyclohexane carboxylate (3a) was chosen as an initial model system for our study. The scope of the study was expanded to include other analogs bearing different substituents on the aromatic moiety (3b−g), and their reactivity and biological activity were compared with those of the model substrate and the natural product aspernomine.
To prepare the starting materials, a Diels−Alder coupling based on existing protocols in the literature was proposed. 11 An effective two-step synthesis for the dienophile involving the aromatic nucleophilic substitution of dimethyl malonate on selected 2-fluoronitrobenzenes followed by treatment of nitromalonates 1a−g with paraformaldehyde, Bu 4 NI, and K 2 CO 3 afforded nitroaryl propenoates 2a−g in high to very high yields. 12 Subsequent coupling of the acrylates with the selected diene and hydrolysis of the silylated products furnished the precursors 3a−g. Following an analogous protocol, acrylate 2a was coupled with Danishefsky's diene 13 to provide the additional precursor 3h in 76% yield with the alkene preinstalled, ready for further elaboration (Scheme 2).
With the precursors in hand, several studies using 3a as the model substrate were carried out to achieve the cyclization product (Table 1). In early attempts employing Bredereck's reagents, the unwanted formylation process 8b,9 was clearly Scheme 1. (a) Proposed Retrosynthesis of Aspernomine; (b, c) (Table 1, entry 3), the reaction provided traces of the desired product and the starting material was largely unconsumed. However, the total isolated amount was poor and contained undetermined impurities. Promoting the cyclization with potassium carbonate and copper iodide proved to be a more efficient strategy, affording the product in 37% yield (Table 1, entry 4). However, despite the existing precedents, 10 copper iodide was ineffective for the targeted intramolecular cyclization (Table 1, entry 5). At this point, the reaction time was significantly reduced by applying microwave irradiation, which also slightly improved the product yields ( Once the reaction conditions for the cyclization of the model substrate 3a had been optimized, we focused on the cyclization of the additional precursors prepared earlier (3b−h) ( Figure 2). In general, modifications of the model substrate resulted in lower but still satisfactory yields. Notably, the presence of electron-withdrawing substituents at the meta position of the aromatic ring as well as the introduction of a double bond, which would be beneficial for further elaboration of the benzazocine framework, had slightly detrimental effects on the reaction. In contrast, the presence of an electron-donating group had a less significant impact on the isolated yields. Among all of the prepared compounds, product 4e was the most crystalline and was submitted to X-ray diffraction to confirm its structure.
It was observed that the synthesized products were prone to partial degradation after prolonged storage. Therefore, with the aim of making these compounds easier to handle, we decided to protect the nitrogen atom (Scheme 3). However, using benzazocine 4a as a test substrate, this task proved to be less straightforward than envisaged due to the poor nucleophilicity of the nitrogen atom caused by the neighboring electronwithdrawing groups. Attempted protection with a range of groups such as methyl chloroformate, p-TsCl, CbzCl, Ac 2 O, and Boc 2 O in the presence of various bases (TEA, DIPEA, K 2 CO 3 , NaOH, and NaH) did not result in any reaction. Interestingly, when the stronger base LiHMDS was used in combination with Boc anhydride, 15 the protection of the nitrogen atom was accompanied by O-tert-butoxycarbonylation of the carbonyl group, providing benzazocine 5 along with recovered starting material. At this point, the formation of acetal 6 was performed to block the α carbon. Subsequent protection of the nitrogen atom under the conditions affording 5 gave compound 7 in excellent 91% yield.
On the basis of the observed reactivity of the different products, a reaction mechanism involving a Grob fragmentation is proposed. 16 After the initial enolate−nitrophenyl coupling, the overall process could imply a ring-opening nucleophilic attack on the carbonyl group of NMP, which would generate the five-atom scaffold required for the concerted fragmentation (Scheme 4). The cleavage of the indicated bonds would cause the reduction of the nitro to a deprotonated hydroxylamine A, which through an iterative sequence would enable a second reduction process, and the resulting amide B could render the targeted benzazocine product by protonation. 17 After optimizing the conditions for the synthesis of the hexahydro-2,6-methano-1-benzazocine scaffold, compounds 4a−h were screened for their cytotoxic activity against the human breast cancer cell line MCF7. As shown in Table 2, the activity of the compounds was generally lower than that of the natural product aspernomine, which indicates that the cytotoxicity may not solely depend on the presence of the benzazocine moiety, and other structural factors in the natural product may also be important. Alternatively, the presence of an ester moiety or the racemic nature of our compounds may have contributed to the lower activities. However, the activity of substrate 4h, which bears an enone fragment, was notably higher compared to the natural product and gave low IC 50 values when tested against a variety of cancer cell lines. It is known that Michael acceptors can act as enzyme inhibitors by irreversibly alkylating cysteine residues via conjugate addition, 18 and the toxicity of these compounds is likely attributable to nonspecific protein aggregation. 19 However, it should also be pointed out that a number of Michael acceptor motif-containing drugs are cutting-edge treatments for several types of cancer, 20 which endorses the biosynthetic interest of this structural framework and its further study.
In conclusion, we have developed efficient access to the important hexahydro-2,6-methano-1-benzazocine framework in only four steps using a Diels−Alder reaction and a novel basemediated nitrophenyl reductive cyclization. The preparation of several analogs and their subsequent biological evaluation revealed that the heterocyclic core is less cytotoxic compared to aspernomine, whereas the introduction of an enone functionality notably increased the antiproliferative activity against human cancer cell lines. Future work will focus on the elaboration of the benzazocine framework toward the total synthesis of aspernomine. ■ EXPERIMENTAL SECTION General Information. All reactions were carried out under an argon atmosphere with dry, freshly distilled solvents under anhydrous conditions. Analytical thin-layer chromatography was performed on SiO 2 (Merck silica gel 60 F 254 ), and the spots were located with 1% aqueous KMnO 4 or 2% ethanolic anisaldehyde. Chromatography refers to flash chromatography and was carried out on SiO 2 (SDS silica gel 60 ACC, 35−75 μm, 230−240 mesh ASTM). Drying of organic extracts during workup of reactions was performed over anhydrous Na 2 SO 4 . Evaporation of solvent was accomplished with a rotatory evaporator. NMR spectra were recorded in CDCl 3 except where stated otherwise, and the chemical shifts of 1 H and 13 C NMR spectra are reported in ppm downfield (δ) from Me 4 Si or referenced at CDCl 3 . All NMR data assignments are supported by gCOSY and gHSQC experiments. Nitrobenzenes used for the reductive cyclization were purchased from Sigma-Aldrich (2,5-difluoronitrobenzene, 2,6-difluoronitrobenzene, and 4-fluoro-3-nitroanisole), Apollo Scientific (5-chloro-2-fluoronitrobenzene and 5-bromo-2-fluoronitrobenzene), Fluka (2-fluoronitrobenzene), and Alfa Aesar (2,4-difluoronitrobenzene). MCF7, SKBR3, MDA-MB-231, 453-WT, MiaPaCa, HeLa, HT29, and PC-3 cell lines were obtained from the cell bank resources from the University of Barcelona. Cells were grown in Ham's F12 medium supplemented with 10% fetal bovine serum (GIBCO, Invitrogen, Barcelona, Spain) and incubated at 37°C in a humidified 5% CO 2 atmosphere. Subculture was performed using 0.05% Trypsin (Merck, Madrid, Spain). For IC 50 determination, cells were plated at a density of 10 000 cells/35 mm diameter wells in 1 mL of medium and let to attach to the dish for 20 h before proceeding to cell incubation. The different compounds were first dissolved in 100% DMSO at 100 mM and then diluted appropriately so that the final concentration of DMSO in cell culture did not exceed 0.1%. Cells were incubated with the compounds at increasing concentrations ranging from 300 nM to 300 μM. After 5 days, viability was determined by the MTT assay. Briefly, culture medium was added with 100 μM sodium succinate plus 0.63 mM 3-(4,5-dimethylthyazol-2-yl)-2,5-diphenyltetrazolium bromide (both from Sigma-Aldrich, Madrid, Spain) and incubated for 3h at 37°C. After incubation, culture medium was removed and lysis solution (0.57% of acetic acid and 10% of sodium dodecyl sulfate in dimethyl sulfoxide) (Sigma-Aldrich, Madrid, Spain) was added. Absorbance was measured at 570 nm in a Varioskan Lux from Thermoscientific using the SkanIt TM software v.6.0 to determine the percentage of cell survival relative to the untreated controls. IC 50 was calculated using the specific application within GraphPath Prism v9.0.1 In addition, cell images for each condition were taken using a ZOE Fluorescent Cell Imager (Bio-Rad Laboratories, Inc., Spain) before the MTT assays.